Alzheimer's Disease is a form of dementia marked by progressive intellectual deterioration without focal, motor or sensory signs. It is an insidious, progressive, debilitating disease of unknown etiology afflicting almost two million Americans, most of them elderly.
Definitive diagnosis of the disease is often difficult since many other disorders and neurological deficits are also accompanied by cognitive loss, and histological examination of brain biopsies is an unacceptable procedure in the majority of cases. Thus, only a very small percentage of the total population of Alzheimer's patients is diagnosed early, and those individuals successfully identified are often well advanced in the disease process. Moreover, multiple psychological and biological tests need to be performed to eliminate other non-Alzheimer's related causes of dementia (NIH Publication No. 84-2251, 1984). Consequently, appropriate treatments are difficult to plan. Determining whether one of the causes of dementia is Alzheimer's requires a tool which unequivocally detects Alzheimer's Disease or, at least, eliminates most of the non-Alzheimer's related causes of dementia.
Although the etiology of the disease is unknown, researchers have identified three histopathological structures which are present in the brains of Alzheimer's patients: neurofibrillary tangles, neuritic (senile) plaques, and cerebrovasculature plaques. Neurofibrillary tangles (NT) are intracellular accumulations of fibrous material in the cell bodies of affected neurons, mainly in the hippocampus, amygdala and neocortex. Neuritic and cerebrovascular plaques are found in highest concentration in the hippocampus and neocortex and result from a pathological deposition of amyloid precursor protein(s) (APP) or fragments thereof in these regions. It should be noted that the term amyloid, as used in a neuropathological context, refers to the deposition of APP and its fragments into plaques. This differs from the usage of the term in the general, histopathological context. Specifically, when applied to neuropathological plaques, the term most commonly refers to the A4 amino acid fragment of APP. In standard histopathological uses the term anyloid refers to a refractive, insoluble, noncellular material.
Formation of plaques is now known to be one of the earliest events in the progression of the disease (Terry et al., J. Neuropathol. Exp. Neurol. 46:262-268 (1987); Wisniewski et al., Banbury Report, Davies and Finch, eds. (Cold Spring Harbor Laboratory, NY, pp. 1-26 (1988))). The clinical significance of plaque formation has been highlighted by quantitative studies showing a significant correlation between the numbers of neuritic plaques and neurofibrillary tangles and the clinical severity of dementia (Roth et al., Nature, pp. 109-110, Vol. 50 (1966) and Blessed et al., Br. J. Psych., pp. 797-805, Vol. 114 (1968)), i.e., the most serious cognitive deficits are correlated with the largest number of plaques. Correlation of cognitive deficits with plaque deposition has led to a concerted effort in recent years to define the plaque constituents and the mechanism(s) of plaque generation.
It has been demonstrated that an insoluble material known as .beta.-amyloid (Selkoe et al. Science 115:1243-1245 (1982)); (Glenner and Wong Biochem. Biophys. Res. Commun. 122:885-890 (1984); Wong et al. Proc. Natl. Acad. Sci. U.S.A. 82:8729-8732 (1985)), .beta.-protein, or A4 (Masters et al. EMBO J. 4:2757 (1985)), which was isolated from the birefringent amyloid core of plaques, is the principle pathological component of Alzheimer's Disease.
In 1984, Glenner and Wong (Biochem. Biophys. Res. Comm. 122:1131-1135 (1984)) isolated and determined the amino acid sequence of a peptidergic component of the cerebrovascular amyloid plaques. Additional work with peptides isolated from neuritic plaques from Alzheimer's and Down's syndrome patients was done because older Down's syndrome patients also develop progressive dementia quite similar to the dementia associated with Alzheimer's disease (Ellis et al. Neurology 24:101-106 (1974), Wong et al., Proc. Nat. Acad. Sci., U.S.A., pp. 8729-8732, Vol. 82 (1985)). These additionally isolated peptides were characterized and all were found to contain an amino acid sequence which was similar or identical to that originally isolated and reported by Glenner.
The amino acid sequence determined by Glenner and Wong has enabled researchers to use standard recombinant DNA procedures to isolate cDNA clones that encode the mRNA for the precursor protein of this .beta.-amyloid peptide (Kang et al. Nature 733 (1987); Tanzi et al. Science 235:877 (1987)); Robakis et al. Proc. Natl. Acad. Sci. U.S.A. 84:4190 (1987)). Additional research has demonstrated that alternate forms of the precursor mRNA and protein exist (Kitaguchi et al. Nature, pp. 530-532, Vol. 331 (1988); Tanzi et al. Nature 331:528-530 (1988); Ponte et al. Nature, pp. 525-527, Vol. 331 (1988)). Based on these cDNA sequences, the amino acid sequences of the entire protein molecule(s) can be predicted from the open reading frame/coding region of the mRNA. FIG. 1 sets forth the predicted amino acid sequence for APP. It was speculated that the protein would be membrane-bound, possibly a receptor molecule (Kang et al. Nature 325:733 (1987)). Additional reports supported the conclusion that the amyloid precursor protein (APP) is a membrane localized protein (Zimmerman et al. EMBO J. 7:367-372 (1988); Allsop et al. Proc. Natl. Acad. Sci., pp. 2790-2794, Vol. 85 (1988)). More recently, an N-terminal fragment has been reported in the soluble fraction from tissue extracts (Palmert et al. cited in Abraham and Potter Bio/Technology 7:147-153 (1989)).
It is also mentioned in Bio/Technology 7:147-153 that protein studies indicated APP is a membrane glycoprotein which can be detected in two forms using immunoblotting--a membrane-bound form which can be stained with antibodies to the N-terminal and C-terminal portions and a soluble form detected only with N-terminal antibodies. It is further suggested that the soluble form lacks the C-terminus, including the .beta.-protein. It is also mentioned that pulse chase experiments showed that the soluble, extracellular form is derived from the membrane-bound form, probably by proteolytic cleavage. Results of protein studies are presented in Table 1 appearing on page 150 of Bio/Technology as discussed above.
McDonald et al., Alzheimer Disease and Associated Disorders, page 186, Vol. 2, No. 3, 1988, have reported the characterization and purification of a protein present in the cerebrospinal fluid and serum of individuals with Alzheimer's Disease using a monoclonal antibody which reacts immunohistochemically with amyloid deposits. Immunoreactive proteins of apparent molecular size of 100 and 95 kDa were detected using this monoclonal antibody.
Selkoe et al., Proc. Natl. Acad. Scil. U.S.A., pp. 7341-7345, Vol. 85 (October 1988), disclose the detection of a group of .about..about.110 to 135 kDa membrane-bound proteins in human brain, nonneural tissues and cultured cells found to contain APP mRNAs. These proteins were detected by immunoblot using antibodies specific to C-terminal peptides of the predicted precursor protein comprising either the 20 C-terminal amino acids (numbers 676-695) or the 15 C-terminal amino acids (numbers 681-695). No 35 kDa protein appears to have been detected. Detection of a soluble form of precursor proteins, sharing at least one epitope with the C-terminus of APP, in cerebrospinal fluid or other bodily fluids was not reported.
European Patent Application Publication No. 285,159, published on Oct. 5, 1988, describes a human amyloid related protein monoclonal antibody. It is mentioned in column 9 at lines 13-24 that this antibody is potentially useful to diagnose Alzheimer's dementia. The amyloid-related protein recognized by this antibody has a molecular size of about 42-45 kilodaltons.
European Patent Application Publication No. 274,826, published on Jul. 20, 1988, describes the generation of antibodies against recombinant Alzheimer's amyloid protein or immunogenic peptides thereof for cerebral fluid or serum diagnosis of Alzheimer's Disease. There is no indication that one soluble form of APP-related proteins sharing at least one epitope with the C-terminus of APP could be detected in bodily fluids.
Pardridge et al., Biochem. and Biophys. Res. Commun., pp. 241-248, Vol. 145, (May 29, 1987), describes the development of a radioimmunoassay to detect amyloid (A4) peptide or its precursor in human serum or cerebrospinal fluid. A high molecular weight immunoreactive substance was detected in human serum and CSF samples. It is stated on page 245 that the immunoreactive substance in serum or CSF is a high molecular weight protein that is highly homologous or, possibly, identical to the A4 peptide precursor, but was not the amyloid peptide, per se. A synthetic peptide corresponding to the first 28 amino acids of the 43 amino acid A4 peptide coupled to bovine thyroglobulin was used to generate the polyclonal antiserum used to detect this protein.
Japanese Patent Application 62 [1987]-267,297, which was published on Nov. 19, 1987, discloses a monoclonal antibody which reacts against senile plaques. It is mentioned on page 15 that it might be useful in early diagnosis of senile dementia Alzheimer's Type by detecting .beta.-amyloid or its precursor protein possibly present in serum or cerebrospinal fluid.